There is a need in clinical cytology to detect, identify and analyze rarely occurring cells in the peripheral blood stream and various bodily fluids for diagnostic and research purposes.
One example of many such known procedures is extraction of rare fetal cells from the maternal blood stream for the purposes of clinical prenatal care. The fetal cells can cross the placental barrier and enter the maternal blood stream, from where they can be extracted by drawing a sample of the maternal blood and screening it for fetal cells of interest. This procedure is impeded by very low concentration of the fetal cells in the maternal blood stream, which is typically on the order of one fetal cell for tens of thousands of maternal blood cells.
Another example of such cytological procedures is detection of cancerous cells, which are disseminated in the blood stream from solid malignant tumors within the body, accessible only through invasive surgical procedures. The detection of occult tumor cells in peripheral blood is of crucial importance for early diagnosis of cancer as well as for cancer therapy monitoring and characterization of cancer type and stage, including remission as well as the potential relapse of the disease. To ensure cancer diagnosis at the earliest possible stage, it is essential to be able to detect very small numbers of tumor cells, e.g. down to the range of one tumor cell in one million to ten million non-tumor cells. To obtain a statistically significant number of cancer cells, it is necessary to sift out up to tens of millions of hematopoetic cells.
The tasks of detection of rarely occurring events on a conventional microscope slide carrier are not limited to the investigation of the blood samples for the above mentioned or similar purposes. The processes of screening of liquid and air-borne samples for bacteria, other pathogens and biological weapon agents can also greatly benefit from the convenience and efficiency of a microscope-based sample preparation and laser fluorescence scanning.
Genetic screening for gene alterations is still another procedure capable of being done with the above system. Examination of trisomy, monosomy, or specific targetable genes or genetic markers of identifiable defects (such as Down's syndrome or Tay-Sachs disease) could likewise benefit.
In spite of the multitude of various potential applications of the above-described development, the body of the current patent application will be focused on the specific implementation of the procedures for the preparation of the blood cell derived samples for the sake of clarity and consistency.
The tasks in the rare cell studies and other above-mentioned similar procedures are very demanding both with respect to the event detection technique and sample preparation methodology. Conventionally the rare cell detection is done by scanning the blood cells deposited on the microscope slide, rather than in the flow, in spite of the high sorting speed provided by flow cytometers. The slide deposition of the cells allows for convenient scanning, detection, identification, imaging and relocation of the rare cells for further analysis, as well as storage and archiving of the samples and information.
The preparation of the tens of millions cell samples on a single microscope slide requires deposition of cells on the microscope slide with very high density, approaching the ultimate limit density, and a high degree of utilization of the slide surface area, or else multiple slide preparations would be required to analyze the single blood sample.
The state of the art in microscope slide based cell preparation involves different types of leukapheresis procedures, most commonly based on centrifugation, which separate white blood cells and, similar to them and inseparable by physical characteristics, rare cells of interest from other constituents of the whole blood, like the red blood cells, platelets and plasma.
Not every one of the existing microscope slide based cell preparation procedures seems to be acceptable for rare cell detection. For example, regular cytospin preparations can result in a loss of many (e.g. up to ⅔) of the cells.
An example of more efficient cell sample preparation for microscopic analysis (“Detection and Analysis of Cancer Cells in Blood and Bone Marrow Using a Rare Event Imaging System”, Clinical Cancer Research, Vol. 6, 434-442, February 2000) relies on the attachment of live cells to specialized adhesive slides (Paul Marienfeld GmbH & Co., KG, Bad Mergentheim, Germany) in three circular wells, 15 mm diameter each. This procedure is complicated not only by the necessity to deposit live cells, but also by the multi-step processes of fluorescent immunocytochemical staining and washing, which all have to be performed on the slide containing the attached cells. It takes half a day to prepare a slide sample following the aforementioned procedure, which is not prone to automation. Additional drawbacks of this procedure are high cost of the custom-designed slides and low (e.g. only 27%) level of utilization of the slide surface area. With moderate average cell density of 200,000 cells/cm2 only about 1 million cells could be analyzed per slide, requiring multiple slide preparations for multi-million cell screening from the single blood sample.
Microscope slide samples prepared by the aforementioned procedure were subsequently analyzed by an automated digital microscope based fluorescent image analysis system. Inherent limitations of a microscope-based system include tiny field of view and a limited brightness mercury light source and a CCD camera requiring long integration time which resulted in slow frame by frame slide scanning speed, which in turn lead to about one hour of scanning time for one slide.
Other approaches utilize extreme spectral brightness of a laser light source to increase fluorescence excitation intensity and high sensitive photo-multiplying tube (PMT) based detectors to accelerate the fluorescence data acquisition rate. Straight forward translational scanning employed, for example, in commercial GenePix Scanner (Axon Instruments, Inc.) encounters inertial limitations to the scanning speed from the bulky translation stages.
The laser excitation beam can be swept by a galvanometer-actuated mirror scanner. (“Detection and Quantification of Small Number of Circulating Tumor Cells in Peripheral Blood using Laser Scanning Cytometer (LSC®)” Clin Chem Lab Med, Vol. 39, 811-817, 2001). This instrument scans the laser spot within the tiny field of view of the microscope objective lens, which produces high spatial resolution, but at the expense of slow overall scanning speed, allowing for the analysis of only 50,000 cells in 30 min.
Significant acceleration of scanning speed was achieved by increasing the galvanometric sweeping range of the excitation laser spot up to the full 50 mm width of the microscope slide (“A Rare-Cell Detector for Cancer”, PNAS, Vol. 101, No. 29, 10501-10504, Jul. 20, 2004). The combination of fast (100 scans per second) galvanometric sweeping and 2 mm/sec translation resulted in surface scanning speed of 1 cm2/sec and allowed to pre-scan 25 cm2 slide area for rarely occurring occult tumor cells at the scan rate of 400,000 peripheral blood cells per second. The fluorescence emission was collected by a straight portion of a linear-to-circular fiber optic aperture transformer placed close to the swept laser spot. The filtering aperture is grossly under-utilized in this case, since only a few of the optical fibers in the linear bundle are collecting the fluorescent light at a time and deliver it to the circular bundle. This drawback implicates the usage of a multi-lens collimating optical system, jeopardizes pass band and stop band characteristics of oversized optical fluorescence emission filters, and requires an oversized PMT photocathode. An additional disadvantage of galvanometric mirror sweeping is deviation from linearity of the combination of galvanometer and F-Theta scanning lens, which produces significant beam position distortion.
To implement a successful, cost effective, and widespread system for detection of rarely occurring cells, there are needs for both the efficient and automatable cell deposition on a microscope slide and a fast, simple, and reliable fluorescence detection technique.